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小鼠IDO真核表达载体的构建及其在未成熟树突状细胞中的表达

Construction of eukaryotic expression vector containing mouse IDO gene and its expression in immature DCs
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摘要 目的构建小鼠pEGFP-N1-IDO基因真核表达载体并观察其在未成熟树突状细胞中的表达。方法利用RT-PCR方法扩增小鼠IDO基因全长,利用DNA重组技术将其定向插入到真核表达载体pEGFP-N1,经酶切和测序鉴定后,用DOTAP脂质体转染法转染未成熟树突状细胞,通过G418筛选,转染的未成熟树突状细胞,用倒置荧光显微镜观察绿色荧光蛋白的表达,用RT-PCR、Western-blot检测IDO的表达。结果小鼠全长IDO基因序列正确插入pEGFP-N1载体,与GenBank中报道的序列一致,成功构建了pEGFP-N1-IDO真核表达载体,并转染未成熟树突状细胞,成功地表达目的基因。结论真核表达载体成功构建和转染未成熟树突状细胞,并证明能有效表达于未成熟树突状细胞中。 Objective To construct a eukaryotic expression vector containing mouse IDO gene fused with enhanced green fluorescent protein(pEGFP-N1) and its expression in immature DCs.Methods The full-length IDO gene was obtained from mouse by RT-PCR and was inserted into eukaryotic expression vector pEGFP-N1,after the identification by digestion and sequencing on the recombinant eukaryotic expression vector pEGFP-N1-IDO,the recombinant was transfected into immature DCs by DOTAP liposome.After screening culture by G418,immature DCs was transfected.The green fluorescence protein expression was confirmed by inverted fluorescence microscopy and the transcription and expression of IDO were identified by RT-PCR,Western blotting.Results The full-length coding sequence of IDO was inserted into eukaryotic expression vector pEGFP-N1,IDO gene was successfully amplified and identified by DNA sequencing.The eukaryotic expression vector pEGFP-N1-IDO was constructed and transfected into immature DCs successfully.The IDO gene was expressed successfully.Conclusion The construction of the eukaryotic expression vector pEGFP-N1-IDO,IDO gene could be expressed efficiently in immature DCs transfected by pEGFP-N1-IDO.
出处 《中华肺部疾病杂志(电子版)》 CAS 2008年第1期87-90,共4页 Chinese Journal of Lung Diseases(Electronic Edition)
基金 国家自然科学基金资助项目(30500231)
关键词 吲哚胺2 3双加氧酶 绿色荧光蛋白 真核表达载体 未成熟树突状细胞 Indoleamine 2 3-dioxygenase Green fluorescent protein Eukaryotic expression vector Immature DC
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参考文献10

  • 1张弘超,梁健.不同方法对未成熟树突状细胞体外诱生的研究[J].中国现代医学杂志,2007,17(22):2728-2731. 被引量:4
  • 2冯剑锷,孙宗全,史嘉玮,高开柱.低剂量粒巨噬细胞集落刺激因子培养小鼠骨髓源性未成熟树突状细胞的实验研究[J].中华实验外科杂志,2006,23(2):221-223. 被引量:13
  • 3Munn DH,Sharma MD,Lee JR,et al.Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase[].Science.2002
  • 4Mellor AL,Munn DH.IDO expression by dendritic cells: tolerance and tryptophan catabolism[].Nature Reviews Immunology.2004
  • 5Bauer T M,Jiga L P,Chuang J J,et al.Studying the immunosuppressive role of indoleamine 2,3-dioxygenase: tryptophan metabolites suppress rat allogeneic T-cell responses in vitro and in vivo[].Transplant International.2005
  • 6Austina C J,,Mizdraka J,Matin A,et al.Optimised expression and purification of recombinant human indoleamine2,3-dioxygenase[].Protein Expression and Purification.2004
  • 7Jung,ID,Lee,CM,Jeong,YI,Lee,JS,Park,WS,Han,J,Park,YM.Differential regulation of indoleamine 2,3-dioxygenase by lipopolysaccharide and interferon gamma in murine bone marrow derived dendritic cells[].FEBS Letters.2007
  • 8Hill M,Tanguy-Royer S,Royer P,et al.IDO expands human CD4+CD25high regulatory T cells by promoting maturation of LPS-treated dendritic cells[].European Journal of Immunology.2007
  • 9von-Rango U.Fetal tolerance in human pregnancy--a crucial balance between acceptance and limitation of trophoblast invasion[].Immunology Letters.2008
  • 10Jones BJ,Brooke G,Atkinson K,et al.Immunosuppression by placental indoleamine2,3-dioxygenase:a role for mesenchymal stem cells[].Placenta.2007

二级参考文献14

  • 1吴舰宇,宋春芳,许评,宋纯,石于波,刘锐.应用骨髓法培养树突状细胞的研究[J].中华实验外科杂志,2005,22(7):796-797. 被引量:6
  • 2刘红耀,宁松毅,庞东梓,李梦强,米振国,史天良,叶章群.一种简便、高效的大鼠树突状细胞的分离方法[J].山西医科大学学报,2005,36(6):656-659. 被引量:1
  • 3Lutz MB,Suri RM,Niimi M Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo.Eur J Immunol,2000,30:1813-1822.
  • 4Lutz MB,Kukutsch N,Ogilvie AL An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.J Immunol Methods,1999,223:77-92.
  • 5Inaba K,Inaba M,Romani N Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.J Exp Med,1992,176:1693-1702.
  • 6Heufler C,Koch F,Stanzl U Interleukin-12 is produced by dendritic cells and mediates T helper 1 devdopment as well as interferon-gamma production by T helper 1 cells.Eur J Immunol,1996,26:659-668.
  • 7Steinbrink K,Wolff M,Jonuleit H Induction of tolerance by IL-10-treated dendritic cells.J Immunol,1997,159:4772-4780.
  • 8Bonham CA,Lu L,Banas RA TGF-beta 1 pretreatment impairs the allostimulatory function of human bone marrow-derived antigenpresenting cells for both naive and primed T cells.Transpl Immunol,1996,4:186-191.
  • 9Caux C,Massacrier C,Vanbervliet B Interleukin 10 inhibits T cell alloreac-tion induced by human dendritic cells.Int Immunol,1994,6:1177-1185.
  • 10Jonuleit H,Schmitt E,Schuler G Induction of interleukin 10-producing,nonproliferating CD4(+) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells.J Exp Med,2000,192:1213-1222.

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